Methods for producing Factor VIII proteins

ABSTRACT

Methods are provided for purification of Factor VIII polypeptides by affinity chromatography and ion exchange chromatography, in which the eluate from the affinity column is diluted with a solution comprising higher salt concentration, or lower non-polar agent concentration than that of the elution solution, prior to passing the diluted solution through the ion exchange column. The affinity matrix may comprise a monoclonal antibody or a peptide ligand. The methods result in improved purification without significant yield loss.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-part of application Ser. No.10/728,242, filed on Dec. 3, 2003, now abandoned, which application is aContinuation of application Ser. No. 09/752,280, filed Dec. 29, 2000,now U.S. Pat. No. 6,683,159, which application is a Continuation ofapplication Ser. No. 09/266,322, filed on Mar. 11, 1999, now abandoned,which application claims priority from Provisional Application No.60/077,802, filed on Mar. 12, 1998.

FIELD OF THE INVENTION

The present invention relates to improved methods for the purificationof procoagulant proteins, particularly recombinant production of FactorVIII and related proteins.

BACKGROUND OF THE INVENTION

Hemophilia is an inherited disease which has been known for centuries,but it is only within the last few decades that it has been possible todifferentiate among the various forms; hemophilia A and hemophilia B.Hemophilia A is caused by strongly decreased level or absence ofbiologically active coagulation factor VIII, which is a protein normallypresent in plasma.

Factor VIII (FVIII) is a large and complex glycoprotein thatparticipates in the blood coagulation cascade. Deficiencies in FVIIIproduction in vivo caused by genetic mutation can cause hemophilia,which is treated by infusion of purified preparations of human FVIII(Lee, Thromb Haemost, 82:516-524, 1999). The first purified FVIIIproducts were derived from human serum, isolated from thecryoprecipitate from the Cohn fractionation process.

Research efforts have focused on the development of methods for creatingand isolating highly purified, biologically active factor VIII infull-length and derivative forms. Advantages of a highly purifiedprotein include reduced levels of extraneous proteins in the therapeuticmix as well as a decreased likelihood of the presence of infectiousagents. A more purified form of factor VIII may also be administered insmaller doses, possibly reducing the risk of developing anti-factor VIIIantibodies, as lower doses would be less challenging to the immunesystem.

Significant steps have been taken toward the recombinant production offactor VIII beginning with the isolation of biologically active factorVIII fragments. See, U.S. Pat. No. 4,749,780; U.S. Pat. No. 4,877,614.The gene encoding the full-length human factor VIII protein was isolatedby taking advantage of its sequence homology with porcine factor VIII.See, U.S. Pat. No. 4,757,006. DNA sequences encoding the humancoagulation cofactor, Factor VIII:C (FVIII), are also known in the art[see, e.g., Toole et al., Nature 312:312-317, 1984; Wood et al., Nature312:330-337, 1984; Vehar et al.; Nature 312:337-342, 1984], as well asmethods for expressing them to produce recombinant FVIII [see e.g.Toole, U.S. Pat. No. 4,757,006; WO 87/04187, WO 88/08035 and WO88/03558]. The expression of human and porcine protein having factorVIII:C procoagulant activity was also described in U.S. Pat. No.4,575,006. Active variants and analogs of FVIII protein, and DNAsequences encoding them, have also been reported [see, e.g. Toole, U.S.Pat. No. 4,868,112; EP 0 786 474; WO 86/06101 and WO 87/07144].Generally, such variants and analogs are modified such that part or allof the B domain is missing and/or specific amino acid positions aremodified, for example, such that normally protease-labile sites areresistant to proteolysis, e.g., by thrombin or activated Protein C.Other analogs include modification at one or more lysine and/or tyrosineresidues.

Since severe side effects involving the production of anti-factor VIIIantibodies exist with the administration of the protein isolated fromboth human and non-human sources, truncated lower molecular weightproteins exhibiting procoagulant activity have been designed. U.S. Pat.No. 4,868,112 reports an alternative method of treatment with lowermolecular weight porcine factor VIII of approximately 2000 amino acidsexhibiting similar procoagulant activity as full-length factor VIII. Theremoval of certain amino acids and up to 19 of the 25 possibleglycosylation sites reduced the antigenicity of the protein and therebythe likelihood of developing anti-factor VIII antibodies. However, onedifficulty with the development of recombinant factor VIII is achievingproduction levels in sufficiently high yields.

Various Factor VIII cDNA molecules coding for recombinant factor VIIIderivatives have been developed. For example, U.S. Pat. No. 5,661,008(“the '008 patent”) describes a modified factor VIII derived from afull-length factor VIII cDNA that, when expressed in animal cells,produced high levels of a factor VIII-like protein with factor VIIIactivity. The protein consisted essentially of two polypeptide chainsderived from human factor VIII, the chains having molecular weights of90 kDa and 80 kDa, respectively. The final active protein is made up ofamino acids 1 to 743 and 1638 through 2332 of human factor VIII, thedescription of which is incorporated by reference herein in itsentirety. This polypeptide sequence is commercially known as rFVIII-SQor REFACTO®. See also, Lind et al., Euro. J. Biochem., 232:19-27 (1995);Sandberg et al., Sem Hematol, 38 (Suppl 4):4-12, 2001.

Other forms of truncated FVIII can also be constructed in which theB-domain is generally deleted. In such embodiments, the amino acids ofthe heavy chain, consisting essentially of amino acids 1 through 740 ofhuman Factor VIII and having a molecular weight of approximately 90 kDare connected to the amino acids of the light chain, consistingessentially of amino acids 1649 to 2332 of human Factor VIII and havinga molecular weight of approximately 80 kD. The heavy and light chainsmay be connected by a linker peptide of from 2 to 15 amino acids, forexample a linker comprising lysine or arginine residues, oralternatively, linked by a metal ion bond.

Affinity chromatography offers a powerful method for proteinpurification, with the potential to provide exquisite selectivity fromcontaminating proteins based on the unique interaction between thetarget protein and ligand immobilized on the resin (Carlsson et al.,Affinity chromatography. In: Protein purification: Principles, highresolution methods, and applications. Editors Janson J-C, and Ryden L,New York, Wiley-Liss p 375-442, 1998; Harakas, Biospecific affinitychromatography. In: Protein purification process engineering. EditorHarrison R G, New York, Marcel Dekker p 259-316, 1994). Development ofan affinity chromatography step can be difficult if a ligand withsuitable affinity or selectivity cannot be identified, or if the elutionof the product cannot be achieved without resorting to extremeconditions that may be harmful to the product. While small chemicalligands can be used for affinity separations, their utility hastraditionally been restricted to cases where they act as a substrateanalog, competitive inhibitor, or co-factor for purification of anenzyme. However, recent work with combinatorial libraries has expandedthe repertoire for small molecule ligands (Lowe, Curr Op Chem Biol,5:248-256, 2001; Morrill, J Chrom B, 774:1-15, 2002). Often, thestrength of this binding interaction is moderate to weak (dissociationconstants in the millimolar to micromolar range).

At the other extreme, monoclonal antibodies (Mabs) are used as affinityligands, and often have very high binding affinities (dissociationconstants in the nanomolar range) that are difficult to disrupt withoutextreme pH or high levels of solvents or chaotropes (Goding, Affinitychromatography. In: Monoclonal antibodies: principles and practice.London: Academic Press, p 327-351, 1986). Affinity peptides can bethought of as intermediate between these two cases, as they offer thepotential for enormous diversity in chemical properties, and henceselectivity. Further, by using combinatorial methods based on eitherbiological or chemical systems to generate large libraries of uniquepeptides (Buettner et al., Int J Peptide Protein Res, 47:70-83, 1996;Kaufman et al., Biotechnol Biogen, 77:278-289, 2002; Ladner, TrendsBiotechnol, 13:426-430, 1995; Sato et al., Biotechnol Prog 18:182-192,2002), sequences may be identified with moderate binding affinities thatare sufficient to capture the product without undue losses, but whichare still capable of eluting the bound protein under mild conditions.

Peptide molecules have also been identified as ligands to be used onaffinity chromatography columns. The identification, isolation andsynthesis of binding peptide molecules capable of binding factor VIIIand/or factor VIII-like polypeptides are disclosed in U.S. Pat. No.6,197,526. A phage display method is disclosed that is useful foridentifying families of polypeptide binding molecules. Using thedisclosed method, several binding peptides exhibiting high affinity forfactor VIII and factor VIII-like peptides were identified and isolated.The identified peptides were shown to bind REFACTO®. The disclosures ofU.S. Pat. No. 6,197,526 are incorporated by reference herein in theirentirety.

In Toole et al., Exploration of Structure-Function Relationships inHuman Factor VIII by Site-Directed Mutagenesis, Cold Spring HarborSymposium on Quantitative Biology, 51:543 (1986) it was reported thatrecombinant FVIII could be purified by a combination of monoclonalantibody or peptide ligand affinity chromatography and ion-exchangechromatography. U.S. Pat. No. 5,470,954 describes a similar process inwhich FVIII is passed directly from immunoaffinity purification to theion exchange column. In that document, it is stated that changing theionic strength or polarity of the eluted polypeptide solution increasesthe chance that monoclonal antibodies will remain bound to the FVIIIpolypeptide and co-purify.

U.S. Pat. No. 6,683,159 describes methods for purification of FactorVIII polypeptides by affinity chromatography and ion exchangechromatography. The disclosed method includes the step of diluting theelution solution with a solution comprising higher ionic strength thanthat of the elution solution, resulting in a diluted Factor VIIIsolution. In an alternate embodiment, an elution solution is used thathas a lower concentration of non-polar agent than that of the desorbingsolution. The methods disclosed therein resulted in improvedpurification without significant yield loss. The disclosure of U.S. Pat.No. 6,683,159 is hereby incorporated by reference in its entirety.

SUMMARY OF THE INVENTION

In the present invention, it has been found that diluting the eluatefrom the monoclonal antibody or peptide ligand column provides certainadvantages in processing time and/or reduced monoclonal antibodycontamination of the FVIII protein being purified therefrom. Inaddition, it has been found that adding a salt to the eluate or thediluted eluate will increase recovery of FVIII protein. Accordingly, thepresent invention provides improved methods for the purification ofprocoagulant proteins, including both FVIII and variants thereof, whichmay be produced by recombinant techniques in higher yield and/or in morehomogeneous form.

The present invention provides improved methods of purification of VIIIprotein from cell cultures, preferably from recombinant cell cultures.The methods provide for obtaining FVIII protein of a higher purity thanmethods currently in use. In one embodiment, the methods of the presentinvention comprise diluting the eluate from the immunoaffinity columnwith a solution of higher ionic strength than the eluate solution. Inanother embodiment, the methods of the present invention comprisediluting the eluate from an affinity column with a solution containinglower amounts of ethylene glycol than contained in the eluate solution.A further embodiment is a method wherein a salt, in particular NaCl, isadded to the eluate or diluted eluate to increase recovery.

DETAILED DESCRIPTION OF THE INVENTION

The original manufacturing process for Factor VIII peptides uses amonoclonal antibody (Mab) affinity step, which provides excellentremoval of process-related impurities such as DNA and host cell proteins(Eriksson et al., Sem Hematol, 38 (Suppl 4): 24-31, 2001). However,replacement of the Mab step with a peptide ligand could conferadditional process benefits, including higher column loading capacities,improved resin cleaning afforded by a broader sanitization solutioncompatibility, and cost reduction in raw materials. Most importantly,however, the peptide can be chemically synthesized, eliminating therequirement for a Mab derived from murine hybridomas. Substitution of achemically-synthesized ligand for the Mab eliminates any potential forintroduction of an adventitious virus into the FVIII product stream,such as infectious retroviruses known to be associated with hybridomacultures (Adamson, Dev Biol Stand 93:89-96, 1998; Bartal et al., MedMicrobiol Immunol [Berl] 174:325-332, 1986; Machala et al. Folia Biol[Praha] 32:187-182, 1986). A virus removal filtration step may also beincorporated into the process to provide the greatest possible degree ofassurance that the VIII product and similarly other peptides havingfactor VIII activity are free of adventitious viral contaminants.

The use of peptide ligands in affinity chromatography of Factor VIII isdescribed in Kelley et al, Biotechnology and Bioengineering,87(3):400-412 (2004), and Kelley et al, Journal of Chromatography A,1038:121-130 (2004), the disclosures of which are incorporated byreference herein in their entirety. As shown in these publications,phage display technology can lead to the rapid identification ofaffinity ligands that are particularly well suited for chromatographicuse. In addition, the peptide can incorporate beneficial designelements, for example, confer resistance to chemicals used for cleaningthe resin, allow for directed immobilization, or provide spacers toovercome steric hindrance.

Accordingly, the present invention provides improved methods forpurification of a Factor VIII polypeptide comprising:

-   -   a) adding a mixture containing Factor VIII polypeptide to be        purified to an affinity matrix which binds by hydrophobic and/or        electrostatic and/or van der Waals attractions to the FVIII        polypeptide;    -   b) eluting the Factor VIII polypeptide from the affinity matrix        with an elution solution that causes desorption of the Factor        VIII polypeptide;    -   c) diluting the elution solution with a solution comprising        higher ionic strength than that of the elution solution,        resulting in a diluted Factor VIII solution;    -   d) passing the diluted Factor VIII solution through an ion        exchange column capable of binding to the Factor VIII        polypeptide, thereby binding the Factor VIII polypeptide while        allowing contaminants to pass through the ion exchange column;        and    -   e) eluting the purified Factor VIII polypeptide from the ion        exchange column.

The elution solution of step (b) may contain no salt, or alternatively,salt may be added to the solution. The dilution of step (c) ispreferably performed using a solution comprising from about 5 to about20 mM NaCl, preferably about 7 to about 20 mM NaCl, most preferablyabout 7 to 15 mM. The eluting solution is preferably diluted withsalt-containing solution from about 3-fold to about 5-fold, mostpreferably about 3-fold. If salt is added in either step (b) or (c), theeluting solution should preferably be diluted in step (c) such that thesalt is present at about 5 to about 20 mM NaCl, preferably about 7 toabout 15 mM NaCl.

In a preferred embodiment, in step (b) of the method of the presentinvention the elution solution of step (b) comprises a non-polar agent.Any non-polar agent may be used, such as ethylene glycol, dioxane,propylene glycol and polyethylene glycol, or any appropriate low ionicstrength, low polarity buffered solution. Preferably, the elutionsolution of step (b) contains ethylene glycol, more preferably about 50%(v/v) ethylene glycol.

Optionally, the elution solution may also contain a salt, e.g., NaCl, ina range of 1-50 mM, preferably 5-30 mM, more preferably 7-20 mM, andmost preferably 10-15 mM. If the elution solution contains about 7-20 mMof salt, the dilution step (c) may be eliminated.

The dilution of step (c) is performed using a solution comprising lessthan about 50% (v/v) ethylene glycol, such that the final concentrationof ethylene glycol is from about 17% to about 33% (v/v). In a preferredembodiment, the elution solution of step (b) contains 50% (v/v) ethyleneglycol, and the dilution of step (c) is performed using a solutioncomprising no ethylene glycol, such that the final concentration ofethylene glycol is from about 17 to about 33% (v/v), most preferablyabout 33% (v/v) ethylene glycol. Preferably, the elution solution isdiluted from about 1.5-fold to about 3-fold, most preferably about 1.5fold, or 2:3.

The Factor VIII polypeptide of the present invention is generallyproduced recombinantly, but may also be purified from plasma. Therecombinant Factor VIII polypeptide may be natural full length FactorVIII polypeptide, or a variant, such as a B-domain deleted variant ofFactor VIII, including the VIII:SQ variant.

In preferred embodiments, the mixtures containing Factor VIIIpolypeptides may also include detergents and/or solvents, such aspolyoxyethyl detergents, including Triton X-100, Tween 80. In addition,the Factor VIII polypeptide containing solution may include bufferingsubstances, such as histidine. Salts, e.g., CaCl₂ and NaCl, may also bepresent.

Standard columns and resins known in the art may be used in the methodsof the invention. Chromatographic columns ranging in diameter of 0.5-2.5cm and about 10 cm in height are particularly useful. The affinitycolumn useful in the present invention may be any industriallyacceptable column and resin to which is adsorbed one or more monoclonalantibody or peptide ligands, which antibodies or ligands are capable ofbinding to a Factor VIII polypeptide and from which the Factor VIIIpolypeptide may later be released using standard methods and reagents.Standard resins known in the art may be used. For example, NHS Sepharose4 FF from Amersham Biosciences (Arlington Heights, Ill.) may be used.

The affinity chromatography resin is produced by a coupling reactionthat employs a commercially-available pre-activated base matrix, and isvalidated with a robust and scalable coupling process. Onto the resin isloaded a molecule that will bind by hydrophobic and/or electrostaticand/or van der Waals attractions to the FVIII polypeptide. Examples ofsuch molecules are monoclonal antibodies (mAb), peptide ligands andtriazine dyes. The molecule is selected under conditions used foraffinity purification of Factor VIII. The columns may be loaded usingart-recognized conditions. For example, a column may preferably beloaded at moderate flowrate (about 60 cm/h) with 10-25 column volumes ofload.

Suitable monoclonal antibodies, for example, are disclosed in Fass etal., Blood, 59:594-600 (1982). This description is incorporated byreference in its entirety.

Suitable peptide ligands, for example, are disclosed in U.S. Pat. Nos.6,197,526 and 6,492,105. The descriptions of these antibodies andligands are incorporated by reference herein in their entirety. Kelleyet al, Biotechnology & Bioengineering, discloses that a TN8.2 peptideligand was identified by screening a bacteriophage display library. Thepeptides were designed to have between seven and ten residues betweencysteine residues that oxidize to form a disulfide bond, and hence aconstrained ring. See, e.g., FIG. 2 of Kelley et al. The peptide ligandscan be immobilized on the columns using amine, hydroxyl, carboxylate orhydrazine groups. Sundaram et al, “Affinity Separation,” inStephanopolous G. (ed.) Biotechnology, vol. 3. Bioprocessing. New York:VCH, p 643-677.

The peptide ligands can be chemically synthesized, identified by phagedisplay techniques. The peptide contains a disulfide bond-constrainedloop that interacts with Factor VIII preferably in a single site on thelight chain. Preferably, the peptide binds to Factor VIII with adissociation constant of about 0.1 to about 10 μM, more preferably about1 μM, both in free solution and when immobilized on a chromatographicresin. Suitable peptides are described, for example, in U.S. Pat. No.6,197,526. The description of the peptides and how to obtain and usesuch peptides is hereby incorporated by reference in its entirety.

The peptide ligand allows a relatively simple substitution of thepeptide affinity resin for the monoclonal antibody resin. See, Kelly etal and Kelley et al, supra. However, it should be noted that Factor VIIImay bind differently to a monoclonal antibody resin than to a peptideligand resin. For example, Factor VIII may bind through the light chainto a monoclonal antibody resin and through the heavy chain to a peptideligand resin.

Examples of triazine dyes that may be used to purify a Factor VIIIpolypeptide include those well known in the art. ProMetic Biosciences,for example, has columns with triazine dyes.

The present invention will now be described in terms of the followingnon-limiting examples.

EXAMPLE 1

Preparation of Recombinant Factor VIII:SQ

The production of recombinant factor VIII:SQ (r-VIII SQ) was essentiallyperformed as described in patent WO-A-9109122. A DHFR deficient CHO cellline (DG44NY) was electroporated with an expression vector containingthe r-VIII SQ gene and an expression vector containing the dihydrofolatereductase gene. The conditioned medium (containing fetal calf serum) wasclarified and then concentrated by tangential flow filtration. Thesolution was loaded onto an SP Sepharose Fast Flow cation exchangeresin, wherein the r-VIII SQ binds selectively to the resin throughelectrostatic forces.

The r-VIII SQ was eluted from the column at elevated ionic strength byflowing elution solution (0.8 M NaCl, 3 mM EDTA, 0.02% (v/v) surfactant[Octoxynol 9], 0.1 MNH₄Ac, 5 mM CaCl₂, 1M Sorbitol, pH 6.3±0.2) and wascollected as a single UV adsorbing peak. The r-VIII SQ was then putthrough a virus inactivation step employing the solvent/detergent methodusing TNBP [Tri-(n-butyl)phosphate] and surfactant [such as Octoxynol 9,Triton X-100].

The r-VIII SQ was next loaded onto an affinity chromatography gel, wherethe ligand is a monoclonal antibody (mAb, named 8A4) directed towardsthe heavy chain of factor VIII. After washing, the factor VIII waseluted with a buffer containing 0.05 M histidine, 0.05 M CaCl₂ and 50%ethylene glycol and 0.02% Octoxynol 9 (Tween), pH 6.6. The mAb eluatewas loaded onto an anion exchange column, Q Sepharose® FF sold byPharmacia AB of Uppsala, Sweden. After washing, the FVIII SQ was elutedwith a Q elution buffer containing 0.05 M histidine, 4 mM CaCl₂ 0.4 MNaCl, pH 6.3.

In order to improve upon the above purification system, several seriesof experiments were conducted to evaluate the effects on FVIII recoveryof (a) dilution; (b) dilution with added NaCl; and (c) dilution withreduced, or with no, ethylene glycol.

Q Equilibration Buffer

The solution used to equilibrate the Q-column (the same as thedesorption buffer of the monoclonal antibody column) prior to loadingonto the ion exchange column comprises approximately the followingcomposition:

-   -   0.05 M histidine    -   0.05 M calcium chloride    -   50% (v/v) ethylene glycol    -   0.02% (v/v) Octoxynol 9 or other surfactant    -   pH 6.6±0.2        Series 1: Dilution with Q Equilibration Buffer

Following affinity purification, the eluate was diluted from about3-fold to about 5-fold with Q-equilibration buffer. In the 3-folddilution, total recovery of FVIII activity was acceptable, thoughreduced, while murine IgG detected in the eluate was very low. At higherdilutions, the loss of yield of FVIII activity was significant.

Series 2: Dilution with Q Equilibration Buffer Containing NaCl

Following affinity purification, the eluate was diluted from about3-fold to about 5-fold with Q-equilibration buffer containing NaCl inthe range of about 7 to about 20 mM. Dilution generally produces asignificant reduction in the amount of murine IgG across the ionexchange column. Surprisingly, the addition of NaCl also increasedrecovery of FVIII activity. This increase in recovery was sufficient tooffset the loss in recovery resulting from dilution. The best resultswere observed in 3-fold to 5-fold dilutions with NaCl in the range ofabout 10 to about 17 mM NaCl. The best recovery yields of FVIII activitywere obtained with dilutions of about 3-fold with about 15 mM NaCl.Dilution with less than about 7 mM NaCl or greater than about 20 mM NaClresulted in a loss of final recovery of FVIII activity.

The conclusion is that addition of about 7 mM to about 20 mM NaCl to theQ Equilibration Buffer used to dilute the affinity eluate restores theloss of yield associated with dilution without NaCl, while alsoproducing beneficial results by reducing the murine antibody detected inthe eluate. In the most preferred embodiment, addition of Q EquilibriumBuffer with about 15 mM NaCl added produced optimal results.

Series 3 and 4: Dilution with Q Equilibration Buffer with No or ReducedEthylene Glycol

Following affinity purification, the eluate was diluted from about1.5-fold to about 3-fold with Q Equilibration Buffer that does notcontain ethylene glycol, resulting in final ethylene glycol contentvarying from about 50% (v/v) in the Q Equilibration Buffer down to aslow as about 17% (v/v) in the 3-fold dilution without ethylene glycol. A1.5-fold dilution without ethylene glycol resulted in about a 33% (v/v)final ethylene glycol concentration. With decreased ethylene glycolconcentration, total recovery of protein increased over comparabledilution with Q Equilibration Buffer containing about 50% (v/v) ethyleneglycol.

EXAMPLE 2

Introduction

A suitable downstream purification process for Factor VIII:SQ asproduced in Example 1 may consist of five chromatographic steps:cationic exchange (SP Sepharose FF), immunoaffinity (mAb Sepharose FF),anionic exchange (Q Sepharose FF), hydrophobic interaction (HIC, butylSepharose FF), and gel permeation chromatography (Superdex 200 pg). Theeluate from the mAb column may be directly loaded onto a Q-Sepharose FFcolumn. A series of loading conditions on Q-Sepharose FF column wasexamined by PPD (in collaboration with P&U, Stockholm) to (i) study theimpact of the loading conditions on the activity recovery and thereduction in mouse IgG and HCP levels in the Q-Sepharose peak pool; and(ii) establish optimal loading conditions on the anion exchanger.Results of this study are summarized in this Example.

Experimental Procedures

Material

Q-Sepharose FF resin was packed in a 79×5 mm ID Pharmacia HR column. Allbuffers employed in this study were prepared by CTS by establishedprocedures. The mAb peak pool from the purification process was obtainedfrozen at −80° C. from P&U, Stockholm (LtE 923). The COBAS assay kit andmega standard was bought from Chromogenix AB, Sweden.

Procedures

Q-Sepharose Scale Down Runs:

The Q-Sepharose FF column was initially equilibrated with 10 CV ofbuffer at a flow rate of 0.5 ml/min. Subsequently, the mAb peak pool wasdiluted with the appropriate dilution buffer and loaded onto theQ-Sepharose FF column at a flow rate of 0.2 ml/min. The total activityunits loaded in all the experiments was 48,350 U/ml of the resin, and isclose to the upper limit specified in the PLA. The activity of the mAbpeak pool used to perform these experiments was 2860 IU/ml. The loadvolume in the 3-fold and 5-fold dilution experiments was, therefore,78.6 mls and 131 mls respectively. Following the load, the column waswashed with 40 CVs of a buffer containing 150 mM NaCl, 4 mM CaCl₂, 50 mMHistidine, pH 6.6, at a flow rate of 0.32 ml/min (wash 2). The boundprotein was then eluted with a buffer containing 400 mM NaCl, 4 mMCaCl₂, 50 mM Histidine, pH 6.3 at a flow rate of 0.05 ml/min. Wash 2 andelution in all the experiments were performed at a flow rate of 0.05ml/min. Wash 2 and elution in all the experiments were performed in thereverse direction. The column effluent during the various operations wascollected and assayed for activity. A 1.6 cv fraction was pooled duringelution beginning at the upward drift in the absorbance at 280 nm and istermed the peak pool. The load and peak pool samples were assayed formouse IgG and HCP levels by performing ELISA (P&U, Stockholm).

Time Course Stability Studies:

The mAB peak pool was diluted different fold with (i) mAb elution bufferand (ii) mAb elution buffer containing 40 mM NaCl, and incubated at roomtemperature. The activity in these samples was then assayed at differenttime points.

Results and Discussions

Time Course Stability Study:

The mAB peak pool was diluted 2-fold, 3-fold and 5-fold with mAB elutionbuffer and incubated at room temperature. The drop in activity of thesesamples was monitored as a function of time. A modest drop of 20% inactivity was observed over the course of 24 hours. The loss in activitywas negligible at the end of 4 hours, and less than 10% at the end of 8hours. Further, percentage drop in activity was observed to beindependent of the extent of dilution of the mAb peak pool and henceindependent of the solution concentration of FVIII in the mAb elutionbuffer. Similar results were obtained upon dilution of the mAb peak poolwith mAb elution buffer containing 40 mM NaCl.

Q-Sepharose Scale Down Experiments:

Results from the scale-down runs of the Q-Sepharose FF column performedwith the mAb peak pool diluted 3-fold and 5-fold with the mAb elutionbuffer are shown in Table 1.

TABLE 1 Dilution with mAb Elution Buffer Load Activity at ChallengeLoading End of Run Flow Dilution (IU/ml Load Time (% of Initial ThroughFold resin) (IU/ml) (hours) Activity) Loss (%) 3 48,350 953 6.55 82.83.6 5 48,350 572 10.9 70.4 3.3 Wash Wash Peak Dilution #1 #2 Pre-Peak(1.6 cv) Post-Peak Total Fold (%) (%) (%) (%) (%) Recovery 3 0.7 0.1<0.1 57.4 3.9 65.8 0.4 0.1 <0.1 41.3 2.5 47.7

The flow-through losses in both cases were approximately 3.5% of theload, while the combined activity losses in the wash and prepeak sampleswere less then 1%. The activity in the 1.6 cv peak pool for the 3- and5-fold dilution experiments were 57.5% and 41.3%, respectively, of theload, while the post-peak accounted for 3.9 and 2.5% of the loadactivity units, respectively. The corresponding values in manufacturingruns, wherein the mAb peak pool was loaded onto the column with nofurther modification of the eluate, was 5% in the flow-through and 70±9%in the peak pool. The other effluent streams have negligible activity.

These results clearly demonstrate that the yield across the Q-SepharoseFF column is sensitive to the extent of dilution of the mAb peak poolprior to loading onto the column, and decreases with increasingdilution. For a fixed number of activity units loaded onto the column,the operating time increases with dilution. As suggested by the timecourse stability data, a drop in yield can therefore be expected athigher load dilutions. Nevertheless, experimentally obtained activityvalues from the scale down runs was significantly lower than supportedby the time course stability data. One possible explanation is that theadsorption of FVIII:SQ onto the Q-Sepharose resin under diluteconditions leads to stronger interaction with the resin and has adenaturing effect on the protein, thereby leading to a lower recoveryupon elution. The yield at higher dilutions could then be improved byattenuating the ‘FVIII:SQ-resin’ interaction during loading. In order totest this hypothesis, subsequent experiments were performed with the mAbpeak pool diluted with mAb elution buffer containing NaCl.

Dilution with mAb Elution Buffer Containing NaCl:

The results from the Q-Sepharose scale down experiments performed usingmAb peak pool diluted with mAb elution buffer containing variousconcentrations of NaCl is shown in Table 2.

TABLE 2a 5-Fold Dilution With mAb Elution Buffer Containing NaCl LoadLoad NaCl Challenge Loading Activity at End Flow Conc. (IU/ml Load Timeof Run (% of Initial Through (mM) resin) (IU/ml) (hours) Activity) Loss(%) 10 48,350 572 10.9 90.3 6.5 10 48,350 572 10.9 85.8 6.8 15 48,350572 10.9 76.4 6.6 20 48,350 572 10.9 73.3 6.9 20 48,350 572 10.9 88.98.5 Load Wash Peak NaCl #1 Wash #2 Pre-Peak (1.6 cv) Pos-Peak TotalConc. (%) (%) (%) (%) (%) Recovery 10 0.8 0.2 <0.1 73.1 1.8 82.4 10 0.90.2 <0.1 71.8 3.4 83.1 15 0.9 0.1 — 61.0 7.3 75.9 20 0.9 0.2 — 49.8 12.470.3 20 1.0 0.2 — 46.3 20.1 76.1

TABLE 2b 3-Fold Dilution With mAb Elution Buffer Containing NaCl LoadActivity Challenge Loading at End of Run Flow Load NaCl (IU/ml Load Time(% of Initial Through Conc. (mM) resin) (IU/ml) (hours) Activity) Loss(%) 7 48,350 953 6.55 94.4 5.5 10 48,350 953 6.55 86.3 6.0 12.5 48,350953 6.55 91.6 9.1 16.7 48,350 953 6.55 82.0 6.5 Load Wash Peak NaCl #1Wash #2 Pre-Peak (1.6 cv) Post-Peak Total Conc. (%) (%) (%) (%) (%)Recovery 7 1.3 0.1 58.3 9.9 75.1 10 1.3 0.1 79.9 3.2 90.5 12.5 2.1 0.658.8 12.0 82.5 16.7 1.7 0.4 59.3 4.7 72.65-Fold Dilution Experiments:

Loading the diluted mAb peak pool under conditions that attenuate the‘FVIII:SQ-resin’ interaction significantly increased the overallactivity recovery across the Q-Sepharose column. A greater fraction ofthis increase in activity was seen in the peak pool for the runsemploying 10 and 15 mM NaCl in the load, suggesting that there exists anoptimal NaCl concentration that leads to a maximum peak activityrecovery.

In the NaCl concentration range of about 7 to 20 mM, the activity lossin the flow through varied between 6.5 and 8.5%. These values are twiceof that seen in the 5-fold dilution run in the absence of NaCl. Thecombined wash and prepeak activity losses in all cases were less than2%. The activity losses in the post-peak pool increases with increasingNaCl concentration and was as high as 20% at an NaCl concentration of 20mM. This is expected since the protein migrates farther down the columnduring loading and subsequently takes longer to elute when the flow isreversed.

3-Fold Dilution:

As in the case of 5-fold dilution, the overall activity recovery andflow-through losses were higher when loaded in the presence of NaCl. Themaximum overall and peak activity recovery was obtained at a NaClconcentration of 15 mM. However, existence of an optimum NaClconcentration is not as evident at this dilution level as it was at5-fold dilution.

Mouse IgG Results:

The mouse IgG data on the peak and post-peak pools for all 3- and 5-folddilution experiments are shown in Table 3:

TABLE 3 Mouse IgG Data from 3-Fold and 5-Fold Dilution ExperimentsDilution Load NaCl IgG Levels in Peak IgG Levels in Fold ConcentrationPool (ng/KIU) Post-Peak Pool (ng/KIU) 3-fold 0 0.8 2.1 7 0.5 2.0 10 0.85.3 12.5 0.7 1.8 16.7 1.2 3.6 5-fold 0 0.5 2.1 10 0.8 3.2 10 0.8 3.6 150.7 2.3 20 0.4 1.5 20 0.6 1.3

The IgG values in the peak pool for the 3-fold dilution runs varied from0.5 to 1.2 ng/KIU for the 5-fold dilution runs. The corresponding valuesin manufacturing runs, wherein the mAb peak pool was loaded onto thecolumn with no further modification of the elute, averaged 2.3 ng/KIU.Thus, dilution of the mAb peak pool with mAb elution buffer, with orwithout NaCl, prior to loading reduced the IgG levels in the Q-Sepharosepeak pool. This effect, beyond the mere dilution of IgG levels, may bethe result of a given association constant for formation of IgG-FVIII:SQcomplex. Thus, lowering the concentrations of the IgG and FVIII:SQlowers the concentration of the complex, thereby allowing greaterremoval of IgG across the ion exchanger. In both the 3-fold and 5-folddilution experiments, no correlation was observed between IgG values inthe Q-Sepharose peak pool and NaCl concentrations in the load. Thus, inthe range of NaCl concentrations employed in these experiments, additionof NaCl does not appear to provide additional reduction in mouse IgGlevels.

Host Cell Protein Results:

The host cell protein data on the peak pool for the 3-fold and 5-folddilution experiments are shown in Table 4:

TABLE 4 Host Cell Protein (HCP) Levels in 3-Fold and 5-Fold DilutionExperiments Dilution Fold Load NaCl Conc (mM) HCP in Peak Pool (ng/KIU 37 10.3 12.5 4.2 5 10 14.1 15 9.9 20 10.9

The corresponding values in manufacturing runs, wherein the mAb peakpool is loaded onto the column with no further modification of theeluate, averaged 20 ng/KIU. These results suggest that the HCP levels inthe peak pool decrease with increasing NaCl concentrations, and areindependent of the extent of dilution. The addition of NaCl mayattenuate the binding of HCP to the resin and, therefore, allow lowerlevels of HCP in the Q-Sepharose peak pool.

Conclusions

Dilution of the mAb peak pool with mAb elution buffer prior to loadingon a Q-Sepharose column significantly decreased the yield across thisstep. The decrease in yield is an increasing function of the extent ofdilution. However, the solution stability of FVIII is independent of theextent of dilution with mAb elution buffer, thereby suggesting thatloading under dilute conditions leads to a stronger ‘VIII-resin’interaction and has a denaturing effect on the protein. Addition ofsodium chloride to the dilution buffer attenuates the ‘FVIII-resin’interaction and increases the yield across the Q-Sepharose column.Increasing the NaCl concentrations, however, increases the flow-throughand post-peak losses, and hence there exists an optimum NaClconcentration at which the yield losses are significantly offset. Theoptimum concentration for the 3-fold and 5-fold dilution runs appears tobe in the 7 to 20 mM concentration, and more particularly about 15 mM.

Diluting the mAb peak pool with mAb elution buffer also reduced the IgGand HCP levels in the Q-Sepharose peak pool. In the concentration rangeof NaCl examined, HCP levels in the Q-Sepharose peak pool decreased withincreasing NaCl concentrations in the load. Overall, a combination ofdilution of the mAb peak pool and adding NaCl at concentrations of 7 to20 mM resulted in improved purification without significant yield loss.

EXAMPLE 3

Introduction

Definition of Solutions and Operating Conditions

The process conditions developed for the TN8.2 Sepharose step were basedon the preexisting immunoaffinity step, with modifications adopted tostreamline the process or improve efficiency. Slight modifications tothe load composition were incorporated based on optimization of theSP-Sepharose elution buffer, which had a lower sodium chlorideconcentration and no longer contained sorbitol (the binding of BDDrFVIIIto TN8.2 is not affected by this small change in salt or the presence ofsorbitol). Column wash volumes were decreased, with no observable effecton product purity.

The load solution containing solvent and detergent from the virusinactivation step is compatible for loading the column, which ispre-equilibrated in a buffer of similar composition. The TN8.2 Sepharosecolumn is loaded at moderate flowrate (approximately 60 cm/hr) with15-25 column volumes of load. The load is immediately followed with awash buffer containing a reduced level of Triton X-100 and no solvent.This wash is followed by a buffer containing 1M NaCl in order todissociate CHO proteins that may be bound through weak electrostaticinteractions to the ligand or product. This wash also serves to removeexcess light chains of BDDrFVIII that are produced during the cellculture process and are not removed prior to this step. In preparationfor elution, the column is washed with a low ionic strength buffer, andthen eluted with 50% ethylene glycol, in the same manner as theimmunoaffinity column. The product elutes as a single peak collectedfrom the start of UV rise for 1.5 column volumes. The column is thencleaned and regenerated using a low pH 6M guanidine HCl solution, andstored in dilute ethanol between runs for microbiological control.

Load Conditions

Load Composition

Like the monoclonal antibody, 8A4, used in the recombinant Factor VIIIpurification process of Example 1, the TN8.2 load composition consistsof the SP Sepharose elution plus the solvent/detergent chemicals of thesubsequent viral inactivation step. The TN8.2 ligand was originallyscreened for REFACTO® Albumin Free (AF) binding under the REFACTO® 8A4mAb buffer conditions.

EXAMPLE 4

Elution Conditions

Buffer Excipients

The elution conditions were examined as a potential source of increasedrecovery. Earlier work had indicated that NaCl might play a role instabilizing F:VIII activity. It was critical that any changes made tothe elution would not affect the downstream Q Sepharose step. As an ionexchanger, it was necessary to keep the ionic strength of the QSepharose load low, and adding too much NaCl could inhibit binding ofREFACTO® AF. A NaCl concentration of 10-20 mM was shown to provide somestabilization of REFACTO® AF in 50% ethylene glycol. 15 mM was chosen asthe experimental TN8.2 elution NaCl concentration, in order to increasethe chances of observing a benefit while still minimally affecting the QSepharose load conditions.

TABLE 5 TN8.2 loaded at 25,000 U/ml and 75,000 U/ml. 10 mL columns,+/−15 mM NaCl in Wash 3 and elution. [NaCl] in Wash 3 & Elution % PeakTotal Conditions (mM) Recovery Recovery 25,000 U/ml load 0 76 78 25,000U/ml load 15 100 102 75,000 U/ml load 0 58 62 75,000 U/ml load 15 99 102Discussion

These conditions were tested using different batches of both REFACTO®and REFACTO® AF, and also run several times as part of a 1/40^(th)-scaletrain. All runs had elution recoveries of over 80%, most were over 90%.This profound increase in elution recovery was a remarkable breakthroughin TN8.2 development. Furthermore, product purity and quality are notcompromised with increased recovery. The DNA and CHO values, as well asfor the 1/40^(th)-scale runs, were as good or better than those achievedon the 8A4 mAb.

Following the foregoing description, the characteristics important forpurification of Factor VIII polypeptides by affinity chromatography andion exchange chromatography can be appreciated. Additional embodimentsof the invention and alternative methods adapted to a particularsolution or feed stream will be evident from studying the foregoingdescription. All such embodiments and obvious alternatives are intendedto be within the scope of this invention as defined by the claims thatfollow.

1. A method for purification of a Factor VIII polypeptide comprising:(a) adding a mixture containing a Factor VIII polypeptide to be purifiedto an affinity matrix which binds the Factor VIII polypeptide byhydrophobic and/or electrostatic and/or van der Waals attractions; (b)eluting the Factor VIII polypeptide from the affinity matrix with anelution solution which desorbs the Factor VIII polypeptide; (c) dilutingthe elution solution about 3-fold to about 5-fold with a solutioncomprising higher salt concentration than that of the elution solution,resulting in a diluted Factor VIII solution comprising about 7 to about20 mM of a salt; (d) passing the diluted Factor VIII solution through anion exchange column which binds the Factor VIII polypeptide, therebyallowing contaminants to pass through the ion exchange column; and (e)eluting the purified factor VIII polypeptide from the ion exchangecolumn.
 2. The method of claim 1, wherein the affinity matrix comprisesa monoclonal antibody or peptide ligand.
 3. The method of claim 2,wherein the affinity matrix comprises a monoclonal antibody.
 4. Themethod of claim 2, wherein the affinity matrix comprises a peptideligand.
 5. The method of claim 4, wherein the peptide ligand is TN 8.2.6. The method of claim 1, wherein a salt is added to the elutionsolution of step (b).
 7. The method of claim 6, wherein the salt isNaCl.
 8. The method of claim 1, wherein a salt is added to the dilutedFactor VIII solution in step (c).
 9. The method of claim 8, wherein thesalt is NaCl.
 10. The method of claim 8, wherein the elution solution ofstep (b) comprises no NaCl salt, and the diluted Factor VIII solution instep (c) comprises about 15 mM NaCl.
 11. The method of claim 1, whereinthe elution solution of step (b) comprises NaCl salt, and the dilutedFactor VIII solution in step (c) comprises about 15 mM NaCl.
 12. Themethod of claim 1, wherein the elution solution is diluted about 3-fold.13. The method of claim 1, wherein the affinity matrix comprisesmonoclonal antibody, peptide ligand or triazine dye.
 14. The method ofclaim 1, wherein the elution solution comprises ethylene glycol.
 15. Themethod of claim 14, wherein the solution used to dilute the elutionsolution in step (c) comprises ethylene glycol.
 16. A method forpurification of a Factor VIII polypeptide comprising: (a) adding amixture containing a Factor VIII polypeptide to be purified to anaffinity matrix which binds the Factor VIII polypeptide by hydrophobicand/or electrostatic and/or van der Waals attractions; (b) eluting theFactor VIII polypeptide from the affinity matrix with an elutionsolution which desorbs the Factor VIII polypeptide, wherein the elutionsolution comprises at least one of ethylene glycol, dioxane, propyleneglycol and polyethylene glycol; (c) diluting the elution solution about1.5-fold to about 3-fold with a solution comprising lower concentrationof the ethylene glycol, dioxane, propylene glycol and/or polyethyleneglycol than that of the elution solution, resulting in a diluted FactorVIII solution comprising about 17% to about 33% (v/v) ethylene glycol,dioxane, propylene glycol and/or polyethylene glycol; (d) passing thediluted Factor VIII solution through an ion exchange column which bindsthe Factor VIII polypeptide, thereby allowing contaminants to passthrough the ion exchange column; and (e) eluting the purified FactorVIII polypeptide from the ion exchange column.
 17. The method of claim16, wherein the affinity matrix comprises a monoclonal antibody orpeptide ligand.
 18. The method of claim 17, wherein the affinity matrixcomprises a monoclonal antibody.
 19. The method of claim 17, wherein theaffinity matrix comprises a peptide ligand.
 20. The method of claim 19,wherein the peptide ligand is TN 8.2.
 21. The method of claim 16,wherein the elution solution of step (b) comprises 50% (v/v) ethyleneglycol, and the dilution of step (c) is performed using a solutioncomprising less than 50% (v/v) ethylene glycol, such that the finalconcentration of ethylene glycol in the diluted factor VIII solution isfrom about 17% to about 33% (v/v).
 22. The method of claim 16, whereinthe elution solution of step (b) contains 50% (v/v) ethylene glycol, andthe dilution of step (c) is performed using a solution comprising noethylene glycol, such that the final concentration of ethylene glycol inthe diluted factor VIII solution is from about 17% to about 33% (v/v).23. The method of claim 16, wherein the elution solution is dilutedabout 1.5-fold.
 24. The method of claim 16, wherein a salt is added tothe elution solution of step (b).
 25. The method of claim 24, whereinthe salt is NaCl.
 26. The method of claim 16, wherein a salt is added tothe diluted Factor VIII solution in step (c).
 27. The method of claim26, wherein the salt is NaCl.
 28. The method of claim 16, wherein theelution solution of step (b) comprises no NaCl salt, and the dilutedFactor VIII solution in step (c) comprises about 15 mM NaCl.
 29. Themethod of claim 16, wherein the elution solution of step (b) comprisesNaCl salt, and the diluted Factor VIII solution in step (c) comprisesabout 15 mM NaCl.
 30. The method of claim 16, wherein the affinitymatrix comprises monoclonal antibody, peptide ligand or triazine dye.31. The method of claim 16, wherein the elution solution comprisesethylene glycol.
 32. The method of claim 31, wherein the solution usedto dilute the elution solution in step (c) comprises ethylene glycol.33. A method for purification of a Factor VIII polypeptide comprising:(a) adding a mixture containing a Factor VIII polypeptide to be purifiedto an affinity matrix which binds to the Factor VIII polypeptide; (b)eluting the Factor VIII polypeptide from the affinity matrix with anelution solution which desorbs the Factor VIII polypeptide, wherein theelution solution comprises at least one of ethylene glycol, dioxane,propylene glycol and polyethylene glycol and a buffer; (c) diluting theelution solution about 1.5-fold to about 3-fold with a solutioncomprising a lower concentration of the ethylene glycol, dioxane,propylene glycol and/or polyethylene glycol than that of the elutionsolution, resulting in a diluted Factor VIII solution comprising about17% to about 33% (v/v) ethylene glycol, dioxane, propylene glycol and/orpolyethylene glycol; (d) passing the diluted Factor VIII solutionthrough an ion exchange column which binds the Factor VIII polypeptide,thereby allowing contaminants to pass through the ion exchange column;and (e) eluting the purified Factor VIII polypeptide from the ionexchange column.
 34. The method of claim 33, wherein the affinity matrixcomprises a monoclonal antibody or peptide ligand.
 35. The method ofclaim 34, wherein the affinity matrix comprises a monoclonal antibody.36. The method of claim 34, wherein the affinity matrix comprises apeptide ligand.
 37. The method of claim 36, wherein the peptide ligandis TN 8.2.
 38. The method of claim 33, wherein the elution solution ofstep (b) comprises no NaCl salt, and the diluted Factor VIII solution instep (c) comprises about 15 mM NaCl.
 39. The method of claim 33, whereinthe elution solution of step (b) comprises NaCl salt, and the dilutedFactor VIII solution in step (c) comprises about 15 mM NaCl.
 40. Themethod of claim 33, wherein the elution solution is diluted about1.5-fold.
 41. The method of claim 33, wherein a salt is added to theelution solution of step (b).
 42. The method of claim 41, wherein thesalt is NaCl.
 43. The method of claim 33, wherein a salt is added to thediluted Factor VIII solution in step (c).
 44. The method of claim 43,wherein the salt is NaCl.
 45. The method of claim 33, wherein theaffinity matrix comprises monoclonal antibody, peptide ligand ortriazine dye.
 46. The method of claim 33, wherein the elution solutioncomprises ethylene glycol.
 47. The method of claim 46, wherein thesolution used to dilute the elution solution in step (c) comprisesethylene glycol.
 48. A method for purification of a Factor VIIIpolypeptide comprising: (a) adding a mixture containing a Factor VIIIpolypeptide to be purified to an affinity matrix which binds the FactorVIII polypeptide by hydrophobic and/or electrostatic and/or van derWaals attractions; (b) eluting the Factor VIII polypeptide from theaffinity matrix with an elution solution which desorbs the Factor VIIIpolypeptide, wherein the elution solution comprises about 7 to about 20mM of salt; (c) passing the elution solution comprising Factor VIIIsolution through an ion exchange column which binds the Factor VIIIpolypeptide, thereby allowing contaminants to pass through the ionexchange column; and (d) eluting the purified factor VIII polypeptidefrom the ion exchange column.
 49. The method of claim 48, wherein theelution solution of step (b) comprises about 15 mM NaCl.
 50. The methodof claim 48, wherein the affinity matrix comprises monoclonal antibody,peptide ligand or triazine dye.
 51. The method of claim 48, wherein theelution solution comprises ethylene glycol.
 52. A method forpurification of a Factor VIII polypeptide comprising: (a) adding amixture containing a Factor VIII polypeptide to be purified to anaffinity matrix which binds the Factor VIII polypeptide by hydrophobicand/or electrostatic and/or van der Waals attractions; (b) eluting theFactor VIII polypeptide from the affinity matrix with an elutionsolution which desorbs the Factor VIII polypeptide, the elution solutioncomprising either a salt at a concentration of about 1 mM to about 25 mMor at least one of ethylene glycol, dioxane, propylene glycol andpolyethylene glycol at a final concentration of about 50% (v/v); (c)diluting the elution solution about 3-fold to about 5-fold with asolution of higher ionic strength than that of the elution solution; (d)passing the diluted Factor VIII solution through an ion exchange columnwhich binds the Factor VIII polypeptide, thereby allowing contaminantsto pass through the ion exchange column; and (e) eluting the purifiedfactor VIII polypeptide from the ion exchange column.
 53. The method ofclaim 52, wherein the diluted Factor VIII solution comprises about 1 toabout 50 mM of salt in step (c).
 54. The method of claim 52, wherein thediluted Factor VIII solution comprises about 7 to about 20 mM of salt instep (c).
 55. The method of claim 52, wherein the affinity matrixcomprises monoclonal antibody, peptide ligand or triazine dye.
 56. Themethod of claim 52, wherein the affinity matrix comprises monoclonalantibody or peptide ligand.
 57. The method of claim 56, wherein theaffinity matrix is a TN 8.2 peptide ligand.
 58. The method of claim 52,wherein the elution solution comprises ethylene glycol.
 59. The methodof claim 58, wherein the solution used to dilute the elution solution instep (c) comprises ethylene glycol.
 60. The method of claim 52, whereinthe elution solution comprises a salt at a concentration of about 5 mMto about 20 mM.
 61. The method of claim 60, wherein the elution solutioncomprises a salt at a concentration of about 5 mM, about 10 mM, about 15mM, or about 20 mM.
 62. The method of claim 52, wherein the finalconcentration of at least one of ethylene glycol, dioxane, propyleneglycol and polyethylene glycol is about 17% to about 33% (v/v) in step(c).